MACROPHAGE INFLAMMATORY PROTEINS-3, -4 AND -1Sg(g)

Abstract: There are disclosed a human macrophage inflammatory protein-3, a human macrophage inflammatory protein-4 and a human macrophage inflammatory protein-1 gamma polypeptides and DNA (RNA) encoding such polypeptides. There is also provided a procedure for producing such polypeptides by recombinant techniques and for producing antibodies against such polypeptides. In the invention there is also provided antagonist/inhibitors against such polypeptides which inhibit the functioning of such polypeptides. Another aspect of the invention provides a combination of the polypeptides of the present invention and a suitable pharmaceutical carrier for providing a therapeutically effective amount of the polypeptides for the treatment of various associated diseases.

Description: MACROPHAGE INFLAMMATORY PROTEXNS-3, -4 and -I7 This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production of such polynucleotides and polypeptides. More particularly, the polypeptides of the present invention are human macrophage inflammatory protein-3 (MIP-3), macrophage inflammatory protein-4 (MIP-4) and macrophage inflammatory protein-l . The invention also relates to inhibiting the action of such polypeptides. Macrophage inflammatory proteins (MIPs) are proteins that are produced by certain mammalian cells, for example, macrophages and lymphocytes, in response to stimuli, such as gram-negative bacteria, lipopolysaccharide and concanavalin A. Thus, the MIP molecules may have diagnostic and therapeutic utility for detecting and treating infections, cancer, inflammation, myelopoietic dysfunction, and auto¬ immune diseases. Murine MIP-1 is a major secreted protein from lipopolysaccharide stimulated RAW 264.7, a murine macrophage tumor cell line. It has been purified and found to consist of two related proteins, MlP-lα and MIP-lj8. Several groups have cloned what are likely to be the human homologs of MlP-lα and MIP-l/S. In all cases, cDNAs -1- SUBSTITUTESHEET(RULE2fi) were isolated from libraries prepared against activated T- cell RNA. The macrophage inflammatory proteins (MlP-lα and MIP-ljS) have been shown to exhibit pro-inflammation properties. MIP- lα can induce migration and activation of human eosinophils and monocytes and induces the chemotaxis of macrophages. In addition, the murine MlP-lα has suppressive effects on human hematopoietic stem cell proliferation, while the murine MIP- lβ can abrogate the inhibitory 'effect of MlP-lα. Finally, MIP-1 proteins can be detected in early wound inflammation cells and have been shown to induce production of IL-1 and IL-6 from wound fibroblaεt cells. MIP-ls are part of the chemokine family which have numerous functions related to inflammation or wound healing, immunoregulation and play active roles in a number of disease conditions. Further, MIP-1 has been found to have hematopoietic potentiating effects. Broxmeyer, H.E., et al., J. Exp. Med., 170:1583-94 (1989) and Broxmeyer, H.E., et al.. Blood, 76:1110-6 (1990) The definition of the bioactivities of MIP-1 has been extensively studied and has utilized native MIP-1 and very recently recombinant MlP-lα and MIP-1/3. Purified native MIP- 1 (comprising MIP-1, MlP-lα and MIP-13 polypeptides) causes acute inflammation when injected either subcutaneously into the footpads of mice or intracisternally into the cerebrospinal fluid of rabbits (Wolpe and Cerami, 1989, FASEB J. 3:2565-73). In addition to these pro-inflammatory properties of MIP-1, which may be direct or indirect, MIP-1 has been recovered during the early inflammatory phases of wound healing in an experimental mouse model employing sterile wound chambers (Fahey, et al., 1990, Cytokine, 2:92). For example, PCT application U.S. 91/06489, filed by Chiron Corporation, discloses a DNA molecule which is active as a template for producing mammalian macrophage inflammatory proteins (MIPs) in yeast. -2- SUBSTITUTESHEET(RULE2β) The murine MlP-lα and MIP-1/3 are distinct but closely related cytokines. Partially purified mixtures of the two proteins affect neutrophil function and cause local inflammation and fever. The particular properties of MlP-lα have been identified as being identical to an inhibitor of haemopoietic stem cell growth. MlP-lα has been expressed in yeast cells and purified to homogeneity. Structural analysis confirmed that MlP-lα has a very similar secondary and tertiary structure to platelet factor 4 and interleukin 8 with which it shares limited sequence homology. MlP-lα has been found to have in vitro stem cell inhibitory properties. It has also been demonstrated that MlP-lα is active in vivo to protect mouse stem cells from subsequent in vitro killing by tritiated thymidine. MlP-lα was also shown to enhance the proliferation of more committed progenitor granulocyte macrophage colony-forming cells in response to granulocyte macrophage colony-stimulating factor. Clemens, J.M., et al., Cytokine, 4:76-82 (1992). MlP-lα is a 6-8 kd, lipopolysaccharide inducible monocyte, neutrophil and eosinophil chemotactic protein. MlP-lα can also induce the migration and activation of human eosinophil granulocytes. MlP-lα may be important in acute and chronic inflammation. The sequential production, source, and in vivo contribution of murine MlP-lα in synchronized schistosoma mansoni during pulmonary granuloma formation has been determined. Lukacs, N.W. , et al., J. Exp. Med. , 177:1551-9 (1993). In accordance with one aspect of the present invention, there are provided novel mature polypeptides which are MIP-3, MIP-4 and MIP-I7, as well as analogs and derivatives thereof. The MIP-3, MIP-4 and MIP-I7 of the present invention are of human origin. In accordance with another aspect of the present invention, there are provided polynucleotides (DNA or RNA) which encode such polypeptides. -3- SUBSTTTUTESHEET(RULE2β) In accordance with yet a further aspect of the present invention, there is provided a process for producing such polypeptides by recombinant techniques. In accordance with yet a further aspect of the present invention, there is provided a process for utilizing such polypeptides, or polynucleotides encoding such polypeptides for therapeutic purposes, for example, immunoregulation including inflammatory activity, hematopoiesis and lymphocyte trafficking, inhibition of bone marrow stem cell colony formation, treatment of psoriasis, solid tumors, and enhancement of host defenses against resistant chronic infection. In accordance with yet a further aspect of the present invention, there is provided an antibody against such polypeptides. In accordance with yet another aspect of the present invention, there are provided antagonist/inhibitors to such polypeptides, which may be used to inhibit the action of such polypeptides, for example, in the treatment of arteriosclerosis, autoimmune and chronic inflammatory and infective diseases, histamine-mediated allergic reactions, hyper-eoεinophilic syndrome, silicosiε, sarcoidosis, inflammatory diseases of the lung, inhibition of IL 1 and TNF, aplastic anaemia, and myelodysplastic syndrome. These and other aspects of the present invention should be apparent to those skilled in the art from the teachings herein. The following drawings are illustrative of embodiments of the invention and are not meant to limit the scope of the invention as encompassed by the claims. FIG. 1 displays the cDNA sequence encoding MIP-3 and the corresponding deduced amino acid sequence. The initial 45 amino acids represents the deduced putative leader sequence. FIG. 2 displays the cDNA sequence encoding MIP-4 and the corresponding deduced amino acid sequence. The initial 19 amino acids represent a putative leader sequence. FIG. 3 corresponds to a portion of the deduced amino acid sequence of MIP-3 in alignment with a portion of MlP-lα. The top sequence is human MIP-3 amino acid sequence and the bottom sequence is human MlP-lα (Human Tonsillar lymphocyte LD78 Beta protein precursor). FIG. 4 displays two amino acid sequences wherein, the top sequence is the human MIP-4 amino acid sequence and the bottom sequence is human MlP-lα (Human Tonsillar lymphocyte LD78 Beta protein precursor) . FIG. 5 shows the protein bands corresponding to the MIP- 4 protein after expression in an E.coli bacterial expression system and purification. FIG. 6 is a northern blot analysis of the MIP-4 which indicates the cells in which this protein is most commonly found. FIG. 7 is a schematic representation of the pQE-9 vector. FIG. 8 displays the cDNA sequence encoding MIP-I7 and the corresponding deduced amino acid sequence. The initial 24 amino acids represent the deduced leader sequence. FIG. 9 displays two partial amino acid sequences of human MIP-1 proteins. The top sequence is human MIP-I7 and the bottom sequence is human MlP-lα [Human Tonsillar lymphocyte LD78 Beta protein precursor]. FIG. 10 shows the protein bands in lane 5 corresponding to the MIP-I7 protein after expression in a bacterial expression system and purification. FIG. 11 shows the protein bands corresponding to COS expression of MIP-I7. FIG. 12 is a northern blot analysis of the MIP-I7 indicating the tissues in which this protein is most commonly found. -5- SUBST1TUTESHEET(RULE26) In accordance with an aspect of the present invention, there are provided isolated nucleic acids (polynucleotides) which encode for the mature polypeptide having the deduced amino acid sequence of Figures 1, 2 and 8 or for the mature MIP-3 polypeptide encoded by the cDNA of the clone(ε) deposited aε ATCC Deposit No. 75676 on February 9, 1994, and for the mature MIP-4 polypeptide encoded by the cDNA of the clone depεoited aε ATCC Depoεit No. 75675 on February 9, 1994 and for the mature MIP-I7 polypeptide encoded by the cDNA of the clone depoεited as ATCC no. 75572, deposited on October 13, 1993. Polynucleotides encoding polypeptides of the present invention are structurally related to the pro-inflammatory supergene "intercrine" which is in the cytokine or chemokine family. Both MIP-3 and MIP-4 are MIP-1 homologues and are more homologous to MlP-lα than to MIP-13. The polynucleotide encoding for MIP-3 was derived from an aortic endothelium cDNA library and contains an open reading frame encoding a polypeptide of 121 amino acid residues, which exhibits significant homology to a number of chemokines. The top match is to the human macrophage inflammatory protein 1 alpha, showing 36% identity and 66% similarity (figure 3). The polynucleotide encoding for MIP-4 was derived from an adult lung cDNA library and contains an open reading frame encoding a polypeptide of 89 amino acid residues, which exhibits significant homology to a number of chemokines. The top match is to the human tonsillar lymphocyte LD78 beta protein, showing 60% identity and 89% similarity (figure 4). Furthermore, the four cysteine residues occurring in all chemokines in a characteriεtic motif are conserved in both clone(s). The fact that the firεt two cyεteine reεidueε in our gene are in adjacent poεitionε claεεifieε it as "C-C" or β subfamily of chemokines. In the other subfamily, the "CXC" or a subfamily, the first two cysteine residues are separated by one amino acid. The polynucleotide encoding from MIP-I7 contains and open reading frame encoding a polypeptide of 93 amino acids, which exhibit significant homology to human macrophage and primatory protein-α, with 48% identity and 72% similarity over a stretch of 80 amino acids and 46% and 67% εimilarity to mouse MIP-ljS over a region of 82 amino acids. Further, the four cysteine residues comprising a characteristic motif are conserved. The polynucleotides of the present invention may be in the form of RNA or in the form of DNA, which DNA includes cDNA, genomic DNA, and synthetic DNA. The DNA may be double- stranded or single-stranded, and if single stranded may be the coding strand or non-coding (anti-sense) strand. The coding sequence which encodes the mature polypeptides may be identical to the coding sequence shown in Figure 1 and 2 or that of the deposited clone(ε) or may be a different coding sequence which coding sequence, as a result of the redundancy or degeneracy of the genetic code, encodes the same, mature polypeptides as the DNA of Figure 1 and 2 or the deposited CDNA. The polynucleotides which encode for the mature polypeptideε of Figures 1, 2 and 8 or for the mature polypeptides encoded by the deposited cDNA may include: only the coding sequence for the mature polypeptide; the coding sequence for the mature polypeptides and additional coding sequence such aε a leader or εecretory εequence or a proprotein εequence; the coding sequence for the mature polypeptides (and optionally additional coding sequence) and non-coding sequence, such as intronε or non-coding sequence 5' and/or 3' of the coding sequence for the mature polypeptides. Thus, the term "polynucleotide encoding a polypeptide" encompasses a polynucleotide which includes only coding sequence for the polypeptide as well as a polynucleotide which includes additional coding and/or non-coding sequence. -7- SUBSΠTUTESHEET(RULE26) The present invention further relates to variants of the hereinabove described polynucleotides which encode for fragments, analogs and derivatives of the polypeptide having the deduced amino acid sequence of Figures 1, 2 and 8 or the polypeptides encoded by the cDNA of the deposited clone(s). The variantε of the polynucleotideε may be a naturally occurring allelic variant of the polynucleotideε or a non- naturally occurring variant of the polynucleotides. Thus, the present invention includes polynucleotides encoding the same mature polypeptideε aε εhown in Figureε 1, 2 and 8 or the same mature polypeptides encoded by the cDNA of the deposited clone(s) aε well aε variantε of εuch polynucleotides which variantε encode for a fragment, derivative or analog of the polypeptideε of Figureε 1, 2 and 8 or the polypeptides encoded by the cDNA of the deposited clone(s). Such nucleotide variants include deletion variantε, substitution variantε and addition or insertion variants. As hereinabove indicated, the polynucleotide may have a coding sequence which is a naturally occurring allelic variant of the coding sequence shown in Figures 1, 2 and 8 or of the coding sequence of the deposited clone(s). As known in the art, an allelic variant is an alternate form of a polynucleotide sequence which may have a substitution, deletion or addition of one or more nucleotideε, which does not substantially alter the function of the encoded polypeptide. The present invention also includes polynucleotides, wherein the coding εequence for the mature polypeptides may be fused in the same reading frame to a polynucleotide sequence which aids in expression and secretion of a polypeptide from a host cell, for example, a leader sequence which functions aε a εecretory sequence for controlling transport of a polypeptide from the cell. The polypeptide having a leader sequence is a preprotein and may have the -8- SUBSTrUTESHEET(RULE26) leader εequence cleaved by the host cell to form the mature form of the polypeptide. The polynucleotides may also encode for a proprotein which is the mature protein plus additional 5' amino acid reεidueε. A mature protein having a proεequence iε a proprotein and iε an inactive form of the protein. Once the proεequence iε cleaved an active mature protein remains. Thus, for example, the polynucleotides of the present invention may encode for a mature protein, or for a protein having a prosequence or for a protein having both a proεequence and a presequence (leader sequence). The polynucleotides of the present invention may also have the coding sequence fused in frame to a marker sequence which allows for purification of the polypeptides of the present invention. The marker εequence may be a hexa- hiεtidine tag εupplied by a pQE-9 vector to provide for purification of the mature polypeptideε fuεed to the marker in the case of a bacterial host, or, for example, the marker sequence may be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells, is used. The HA tag correεponds to an epitope derived from the influenza hemagglutinin protein (Wilson, I., et al.. Cell, 37:767 (1984)). The preεent invention further relates to polynucleotides which hybridize to the hereinabove-described sequenceε if there iε at least 50% and preferably 70% identity between the sequenceε. The preεent invention particularly relateε to polynucleotides which hybridize under stringent conditions to the hereinabove-described polynucleotides . As herein used, the term "stringent conditionε" means hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequences. The polynucleotides which hybridize to the hereinabove deεcribed polynucleotideε in a preferred embodiment encode polypeptideε which retain subεtantially the same biological function or activity as the mature -9- SUBST1TUTESHEET(RULE26) polypeptide encoded by the cDNA of Figure 1 or the deposited CDNA. The deposit(ε) referred to herein will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Micro-organisms for purposeε of Patent Procedure. Theεe depoεits are provided merely as convenience to those of έkill in the art and are not an admiεεion that a depoεit is required under 35 U.S.C. §112. The sequence of the polynucleotideε contained in the deposited materials, as well as the amino acid sequence of the polypeptides encoded thereby, are incorporated herein by reference and are controlling in the event of any conflict with description of sequences herein. A license may be required to make, use or εell the depoεited materialε, and no such license is hereby granted. The present invention further relateε to MIP-3, MIP-4 and MIP-I7 polypeptideε which have the deduced amino acid sequence of Figures 1, 2 and 8 or which have the amino acid sequence encoded by the depoεited cDNA, aε well aε fragments, analogε and derivativeε of such polypeptide. The terms "fragment," "derivative" and "analog" when referring to the polypeptides of Figures 1, 2 and 8 or that encoded by the deposited cDNA, means a polypeptide which retainε eεεentially the same biological function or activity as such polypeptide. Thus, an analog includes a proprotein which can be activated by cleavage of the proprotein portion to produce an active mature polypeptide. The polypeptides of the preεent invention may be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide, preferably a recombinant polypeptide. The fragment, derivative or analog of the polypeptides of Figures 1, 2 and 8 or that encoded by the deposited cDNA may be (.i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such subεtituted amino acid reεidue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid reεidueε includeε a εubεtituent group, or (iii) one in which the mature polypeptideε are fused with another compound, such aε a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the mature polypeptides, such as a leader or secretory sequence or a sequence which is employed for purification of the mature polypeptides or a proprotein sequence. Such fragments, derivativeε and analogε are deemed to be within the εcope of thoεe skilled in the art from the teachings herein. The polypeptides of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity. The term "isolated" means that the material is removed from its original environment (e.g., the natural environment if it iε naturally occurring) . For example, a naturally- occurring polynucleotideε or polypeptideε present in a living animal iε not isolated, but the same polynucleotides or DNA or polypeptides, separated from some or all of the coexisting materials in the natural system, is isolated. Such polynucleotideε could be part of a vector and/or εuch polynucleotideε or polypeptideε could be part of a compoεition, and εtill be iεolated in that εuch vector or compoεition iε not part of its natural environment. The present invention alεo relateε to vectors which include polynucleotides of the preεent invention, host cells which are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant techniques. Host cellε are genetically engineered (transduced or transformed or transfected) with the vectors of this invention which may be, for example, a cloning vector or an expression vector. The vector may be, for example, in the form of a plasmid, a viral particle, a phage, etc. The engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the MIP-3, MIP-4 and MIP-I7 geneε. The culture conditionε, εuch aε temperature, pH and the like, are those previously used with the host cell selected for expresεion, and will be apparent to the ordinarily εkilled artiεan. The polynucleotides of the present invention may be employed for producing polypeptideε by recombinant techniques. Thuε, for example, the polynucleotide sequence may be included in any one of a variety of expresεion vehicles, in particular vectorε or plaεmids for expresεing a polypeptide. Such vectorε include chromosomal, nonchromosomal and synthetic DNA sequenceε, e.g., derivatives of SV40; bacterial plasmids; phage DNA; yeast plasmidε; vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabieε. However, any other plaεmid or vector may be uεed aε long they are replicable and viable in the host. The appropriate DNA sequence may be inεerted into the vector by a variety of procedures. In general, the DNA sequence is inserted into an appropriate restriction endonuclease site(ε) by procedures known in the art. Such procedureε and others are deemed to be within the scope of those εkilled in the art. The DNA εequence in the expreεεion vector iε operatively linked to an appropriate expression control sequence(s) (promoter) to direct mRNA synthesis. As representative examples of such promoters, there may be mentioned: LTR or SV40 promoter, the E. coli. lac or trp, the phage lambda P L promoter and other pro oterε known to control expresεion of geneε in prokaryotic or eukaryotic cells or their viruses. -12- SUBSΠTUTESHEET(RULE26) The expreεεion vector also contains a ribosome binding εite for tranεlation initiation and a tranεcription terminator. The vector may also include appropriate sequenceε for amplifying expression. In addition, the expression vectors preferably contain a gene to provide a phenotypic trait for selection of transformed host cellε such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such aε tetracycline or ampicillin resistance in E. coli. The vector containing the appropriate DNA sequence as hereinabove described, as well as an appropriate promoter or control sequence, may be employed to tranεform an appropriate hoεt to permit the hoεt to expreεε the protein. Aε representative examples of appropriate hosts, there may be mentioned: bacterial cellε, such as E. coli. Streptomyces. Salmonella Typhimurium: fungal cellε, εuch aε yeaεt; inεect cells such aε Drosophila and Sf9: animal cells such as CHO, COS or Boweε melanoma; plant cellε, etc. The εelection of an appropriate hoεt is deemed to be within the scope of those skilled in the art from the teachingε herein. More particularly, the present invention also includes recombinant constructε comprising one or more of the εequenceε as broadly described above. The constructs comprise a vector, such as a plaεmid or viral vector, into which a sequence of the invention has been inεerted, in a forward or reverse orientation. In a preferred aspect of this embodiment, the construct further comprises regulatory sequenceε, including, for example, a promoter, operably linked to the sequence. Large numbers of suitable vectors and promoters are known to those of skill in the art, and are commercially available. The following vectors are provided by way of example. Bacterial: pQE70, pQE60, pQE-9 (Qiagen), pbs, pDIO, phagescript, psiX174, pbluescript SK, pbskε, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene); ptrc99a, pKK223- 3, pKK233-3, pDR540, pRIT5 (Pharmacia). Eukaryotic: pWLNEO, -13- SUBST1TUTESHEET(RULE26) pSV2CAT, pOG44, pXTl, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any other plasmid or vector may be used as long as they are replicable and viable in the host. Promoter regions can be εelected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers. Two appropriate vectors are PKK232-8 and PCM7. Particular named bacterial promoterε include lad, lacZ, T3, T7, gpt, lambda P R , P L and trp. Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter iε well within the level of ordinary εkill in the art. In a further embodiment, the present invention relates to hoεt cellε containing the above-deεcribed conεtruct. The host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the hoεt cell can be a prokaryotic cell, εuch aε a bacterial cell. Introduction of the conεtruct into the hoεt cell can be effected by calcium phosphate transfection, DEAE- Dextran mediated transfection, or electroporation. (Davis, L., Dibner, M. , Battey, I., Basic Methods in Molecular Biology, (1986)). The conεtructε in hoεt cellε can be uεed in a conventional manner to produce the gene product encoded by the recombinant εequence. Alternatively, the polypeptideε of the invention can be εynthetically produced by conventional peptide εyntheεizerε. Mature proteinε can be expressed in mammalian cells, yeast, bacteria, or other cellε under the control of appropriate promoterε . Cell-free translation systems can also be employed to produce εuch proteinε uεing RNAs derived from the DNA constructε of the preεent invention. Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), the disclosure of which is hereby incorporated by reference. Transcription of the DNA encoding the polypeptides of the present invention by higher eukaryotes is increased by inserting an enhancer εequence into the vector. Enhancerε are ciε-acting elementε of DNA, uεually about from 10 to 300 bp that act on a promoter to increaεe itε tranεcription. Examples including the SV40 enhancer on the late side of the replication origin bp 100 to 270, a cytomegaloviruε early promoter enhancer, the polyoma enhancer on the late εide of the replication origin, and adenoviruε enhancerε. Generally, recombinant expreεεion vectorε will include originε of replication and εelectable markerε permitting transformation of the host cell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiae TRP1 gene, and a promoter derived from a highly-expresεed gene to direct tranεcription of a downεtream εtructural εequence. Such promoterε can be derived from operonε encoding glycolytic enzymes such as 3-phoεphoglycerate kinaεe (PGK), α-factor, acid phosphataεe, or heat εhock proteinε, among otherε. The heterologouε εtructural sequence is aεεembled in appropriate phaεe with tranεlation initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein into the periplasmic space or extracellular medium. Optionally, the heterologouε sequence can encode a fusion protein including an N-terminal identification peptide imparting desired characteristicε, e.g., εtabilization or simplified purification of expressed recombinant product. Useful expression vectors for bacterial use are conεtructed by inεerting a structural DNA sequence encoding a desired protein together with εuitable translation initiation and termination signals in operable reading phase -15- SUBST1TUTESHEET(RULE26) with a functional promoter. The vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the host. Suitable prokaryotic hosts for transformation include E. coli. Bacillus subtilis. Salmonella typhimurium and various specieε within the genera Pεeudomonas, Streptomyceε, and Staphylococcuε, although otherε may also be employed as a matter of choice. As a representative but nonlimiting example, useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmidε compriεing genetic elements of the well known cloning vector pBR322 (ATCC 37017). Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, WI, USA). These pBR322 "backbone" sections are combined with an appropriate promoter and the εtructural εequence to be expressed. F o l l o w i n g transformation of a suitable hoεt strain and growth of the host strain to an appropriate cell density, the εelected promoter is induced by appropriate means (e.g., temperature shift or chemical induction) and cellε are cultured for an additional period. Cellε are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification. Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lyεing agentε, εuch methodε are well known to those skilled in the art. Various mammalian cell culture εyεtemε can also be employed to expreεs recombinant protein. Exampleε of mammalian expreεεion systems include the COS-7 lines of monkey kidney fibroblaεts, described by Gluzman, Cell, 23:175 (1981), and other cell lines capable of expressing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines. Mammalian expression vectorε will comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking nontranscribed sequenceε. DNA εequenceε derived from the SV40 εplice, and polyadenylation εiteε may be uεed to provide the required nontranεcribed genetic elementε. MIP-3, MIP-4 and MIP-I7 are recovered and purified from recombinant cell cultureε by methodε including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography hydroxylapatite chromatography and lectin chromatography. It is preferred to have low concentrations (approximately 0.15-5 mM) of calcium ion present during purification. (Price et al., J. Biol. Chem., 244:917 (1969)). Protein refolding steps can be used, as necessary, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification εtepε. The polypeptideε of the preεent invention may be a naturally purified product, or a product of chemical εynthetic procedureε, or produced by recombinant techniqueε from a prokaryotic or eukaryotic hoεt (for example, by bacterial, yeast, higher plant, insect and mammalian cells in culture) . Depending upon the hoεt employed in a recombinant production procedure, the polypeptideε of the present invention may be glycosylated with mammalian or other eukaryotic carbohydrateε or may be non-glycosylated. Polypeptides of the invention may also include an initial methionine amino acid residue. -17- SUBST1TUTESHEET(RULE26) The polypeptides of the present invention may be used in a variety of immunoregulatory and inflammatory functions and also in a number of diεeaεe conditionε. MIP-3, MIP-4 and MIP-I7 are in the chemokine family and therefore they are a chemo-attractant for leukocyteε (εuch aε monocytes, neutrophils, T lymphocytes, eosinophils, basophilε, etc.). Accordingly, MIP-3, MIP-4 and MIP-I7 can be used to facilitate wound healing by controlling infiltration of target immune cells to the wound area. In a similar fashion, the polypeptides of the present invention can enhance host defenseε againεt chronic infections, e.g., mycobacterial, via the attraction and activation of microbicidal leukocytes. Further, the polypeptides of the present invention may be useful in anti-tumor therapy since there is evidence that chemokine expreεεing cellε injected into tumorε have caused regresεion of the tumor, for example, in the treatment of Kapoεi εarcoma. Also, MIP-3, MIP-4 and MIP-I7 stimulate the invaεion and activation of hoεt defenεe (tumoricidal) cellε, e.g., cytotoxic T-cells and macrophages, and in this way may also be used to treat solid tumors. The polypeptideε may also be useful to inhibit bone marrow stem cell colony formation for leukemia and as adjunct protective treatment of hematopoietic stem cells during cancer chemotherapy. Another use of the polypeptides is the inhibition of T- cell proliferation via inhibition of IL-2 biosyntheεiε, for example, in auto-immune disease and lymphocytic leukemia. MIP-3, MIP-4 and MIP-I7 may also be useful for inhibiting epidermal keratinocyte proliferation which has utility in pεoriasis (keratinocyte hyper-proliferation) since Langerhans cellε in εkin have been found to produce MlP-lα. MIP-3, MIP-4 and MIP-I7 may be uεed to prevent prevent scarring during wound healing both via the recruitment of debris-cleaning and connective tissue-promoting inflammatory cells and by its possible control of excessive TGF3-mediated -18- SUBSΠTUTESHEET(RULE26) fibrosis, in addition these polypeptides may be used to treat stroke, thrombocytosis, pulmonary emboli and myeloproliferative diεorderε, since MIP-3, MIP-4 and MIP-I7 increase vascular permeability. The polypeptides may also be employed in accordance with the present invention by expresεion of εuch polypeptides in vivo , which iε often referred to aε "gene therapy." Thuε, for example, cellε from a patient may be engineered with a polynucleotide (DNA or RNA) encoding a polypeptide ex vivo , with the engineered cells then being provided to a patient to be treated with the polypeptideε. Such methodε are well-known in the art. For example, cellε may be engineered by procedureε known in the art by use of a retroviral particle containing RNA encoding the polypeptides of the present invention. Similarly, cellε may be engineered in vivo for expreεεion of a polypeptideε in vivo by, for example, procedures known in the art. As known in the art, a producer cell for producing a retroviral particle containing RNA encoding the polypeptides of the present invention may be administered to a patient for engineering the cells in vivo and expression of the polypeptides in vivo . These and other methods for administering polypeptideε of the present invention by εuch method should be apparent to those skilled in the art from the teachingε of the preεent invention. For example, the expreεsion vehicle for engineering cells may be other than a retrovirus, for example, an adenovirus which may be used to engineer cells in vivo after combination with a suitable delivery vehicle. The polypeptides of the present invention may be employed in combination with a suitable pharmaceutical carrier. Such compositions comprise a therapeutically effective amount of the protein, and a pharmaceutically acceptable carrier or excipient. Such a carrier includes but is not limited to saline, buffered saline, dextrose, water. -19- SUBST1TUTESHEET(RULE26) glycerol, ethanol, and combinations thereof. The formulation should suit the mode of administration. The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositionε of the invention. Associated with such container(s) can be a notice in the form preεcribed by a governmental agency regulating the manufacture, uεe or εale of pharmaceuticalε or biological products, which notice reflects approval by the agency of manufacture, use or εale for human administration. In addition, the polypeptides of the present invention may be employed in conjunction with other therapeutic compounds. The pharmaceutical compositionε may be adminiεtered in a convenient manner εuch as by the oral, intravenouε, intraperitoneal, intramuεcular, εubcutaneouε, intranaεal or intradermal routes. The amounts and dosage regimenε of MIP- 3, MIP-4 and MIP-I7 adminiεtered to a εubject will depend on a number of factorε εuch as the mode of administration, the nature of the condition being treated and the judgment of the preεcribing phyεician. Generally εpeaking, they are given, for example, in therapeutically effective doses of at least about 10 μg/Kg body weight and in most cases they will be administered in an amount not in excess of about mg/Kg body weight per day and preferably the dosage iε from about 10 μg/Kg body weight daily, taking into account the routes of administration, symptomε, etc. The εequenceε of the preεent invention are alεo valuable for chromosome identification. The εequence iε εpecifically targeted to and can hybridize with a particular location on an individual human chromoεome. Moreover, there is a current need for identifying particular siteε on the chromoεome. Few chromosome marking reagents baεed on actual sequence data (repeat polymorphisms) are presently available for marking chromosomal location. The mapping of DNA to chromosomes according to the present invention is an important first step in correlating those εequenceε with geneε aεεociated with disease. Briefly, sequences can be mapped to chromosomeε by preparing PCR primerε (preferably 15-25 bp) from the cDNA. Computer analyεiε of the cDNA iε used to rapidly select primerε that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the primer will yield an amplified fragment. PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular DNA to a particular chromosome. Using the present invention with the same oligonucleotide primerε, εublocalization can be achieved with panelε of fragmentε from εpecific chromosomes or pools of large genomic clones in an analogous manner. Other mapping strategies that can similarly be uεed to map to itε chromosome include in situ hybridization, prescreening with labeled flow-sorted chromosomeε and preεelection by hybridization to construct chromosome specific-cDNA libraries. Fluorescence in situ hybridization (FISH) of a cDNA cloneε to a metaphaεe chromoεomal εpread can be uεed to provide a preciεe chromosomal location in one step. Thiε technique can be uεed with cDNA aε εhort as 500 or 600 baseε; however, clones larger than 2,000 bp have a higher likelihood of binding to a unique chromosomal location with sufficient signal intensity for simple detection. FISH requires use of the clones from which the EST waε derived, and the longer the better. For example, 2,000 bp is good, 4,000 is better, and more than 4,000 is probably not necessary to get good results a reasonable percentage of the time. For a review of this technique, see Verma et al. , Human Chromosomeε: a Manual of Baεic Techniques, Pergamon Presε, New York (1988). Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found, for example, in V. McKuεick, Mendelian Inheritance in Man (available on line through Johns Hopkins Univerεity Welch Medical Library) . The relationεhip between geneε and diεeaεeε that have been mapped to the same chromosomal region are then identified through linkage analyεiε (coinheritance of phyεically adjacent geneε). Next, it iε neceεεary to determine the differenceε in the cDNA or genomic εequence between affected and unaffected individuals. If a mutation iε observed in some or all of the affected individuals but not in any normal individualε, then the mutation is likely to be the causative agent of the diεeaεe. With current resolution of phyεical mapping and genetic mapping techniques, a cDNA preciεely localized to a chromoεomal region aεεociated with the disease could be one of between 50 and 500 potential causative geneε. (Thiε assumes 1 megabase mapping reεolution and one gene per 20 kb) . Compariεon of affected and unaffected individualε generally involves first looking for εtructural alterationε in the chromoεomeε, εuch as deletions or translocationε that are viεible from chromoεome spreads or detectable using PCR baεed on that cDNA εequence. Ultimately, complete sequencing of geneε from several individualε is required to confirm the presence of a mutation and to distinguiεh mutationε from polymorphiεmε. The present invention is further directed to inhibiting MIP-3, MIP-4 and MIP-I7 in vivo by the use of antiεenεe technology. Antiεenεe technology can be uεed to control gene expreεsion through triple-helix formation or antisenεe DNA or RNA, both of which methodε are based on binding of a polynucleotide to DNA or RNA. For example, the 5' coding -22- SUBSΠTUTESHEET(RULE26) portion of the polynucleotide εequence, which encodeε for the polypeptideε of the preεent invention, iε used to deεign an antiεenεe RNA oligonucleotide of from about 10 to 40 base pairs in length. A DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription (triple helix -see Lee et al., Nucl. Acids Reε., 6:3073 (1979); Cooney et al. Science, 241:456 (1988); and Dervan et al.. Science, 251: 1360 (1991)), thereby preventing tranεcription and the production of MIP-3, MIP-4 and MIP-I7. The antiεenεe RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into the MIP-3, MIP-4 and MIP-I7 (antisense - Okano, J. Neurochem., 56:560 (1991); Oligodeoxynucleotides as Antiεense Inhibitors of Gene Expreεεion, CRC Preεε, Boca Raton, FL (1988) ) . Alternatively, the oligonucleotides described above can be delivered to cells by procedures in the art such that the antisenεe RNA or DNA may be expreεεed in vivo to inhibit production of MIP-3, MIP-4 and MIP-I7 in the manner deεcribed above. Accordingly, antiεenεe conεtructs to the MIP-3, MIP-4 and MIP-I7 can be used to treat disorders which are either MlP-induced or enhanced, for example, atherosclerosiε, auto¬ immune, e.g. multiple εclerosis and insulin-dependent diabetes, and chronic inflammatory and infective diseases, histamine-mediated allergic reactions, rheumatoid arthritiε, εilicoεiε, εarcoidoεiε, idiopathic pulmonary fibroεis and other chronic inflammatory diseases of the lung, idiopathic hyper-eosinophilic εyndrome, endotoxic εhock, histamine- mediated allergic reactions, prostaglandin-independent fever, and aplastic anemia and other cases of bone marrow failure. The polypeptides, their fragments or other derivatives, or analogs thereof, or cells expresεing them can be uεed aε an immunogen to produce antibodieε thereto. Theεe antibodies can be, for example, polyclonal or monoclonal antibodieε. The -23- SUBST1TUTESHEET(RULE26) preεent invention alεo includes chimeric, εingle chain and humanized antibodieε, as well as Fab fragments, or the product of an Fab expresεion library. Various procedures known in the art may be used for the production of such antibodies and fragments. Antibodieε generated against the polypeptides correεponding to a εequence of the present invention or its in vivo receptor can be obtained by direct injection of the polypeptides into an animal or by administering the polypeptides to an animal, preferably a nonhuman. The antibody so obtained will then bind the polypeptides itself. In this manner, even a sequence encoding only a fragment of the polypeptides can be used to generate antibodies binding the whole native polypeptideε. Such antibodieε can then be uεed to iεolate the polypeptides from tisεue expreεεing that polypeptide. For preparation of monoclonal antibodieε, any technique which provideε antibodieε produced by continuouε cell line cultureε can be used. Examples include the hybridoma technique (Kohler and Milstein, 1975, Nature, 256:495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), and the EBV- hybridoma technique to produce human monoclonal antibodieε (Cole, et al., 1985, in Monoclonal Antibodieε and Cancer Therapy, Alan R. Liεε, Inc., pp. 77-96). Techniqueε described for the production of εingle chain antibodieε (U.S. Patent 4,946,778) can be adapted to produce single chain antibodies to immunogenic polypeptideε productε of thiε invention. The preεent invention iε alεo directed to antagoniεt/inhibitorε of the polypeptideε of the present invention, which inhibit or eliminate the function of the polypeptide. Thus, for example, antagoniεtε bind to a polypeptide of the present invention and inhibit or eliminate its function. -24- SUBST1TUTESHEET(RULE26) The antagoniεt, for example, could be an antibody againεt the polypeptide which bindε to the polypeptide or, in εome cases, an oligonucleotide. An example of an inhibitor is a small molecule which binds to and occupies the catalytic site of the polypeptide thereby making the catalytic site inaccessible to subεtrate εuch that normal biological activity iε prevented. Examples of small molecules include but are not limited to small peptides or peptide-like molecules. Alternatively, antagonists to the polypeptides of the present invention may be employed which bind to the receptors to which a polypeptide of the present invention normally binds. The antagonists may be closely related proteins such that they recognize and bind to the receptor siteε of the natural protein, however, they are inactive formε of the polypeptide and thereby prevent the action of MIP-3, MIP-4 and MIP-I7 since receptor sites are occupied. In these ways, the antagonist/inhibitors may be used as an anti-inflammation drug, by preventing the attraction and activation of leukocytes. They may alεo be usef l for inhibiting the chemotaxis and activation of macrophages and their precursors, and of neutrophilε, basophilε, B lymphocytes and some T-cell εubεetε, in auto-immune and chronic inflammatory and infective diseases; inhibiting MIP- induced mast cell and basophil degranulation and, hence, reduction in histamine-mediated allergic reactions; inhibiting deleterious cascades attributed primarily to IL-1 and TNF whose biosynthesis iε stimulated by MIPs; rheumatoid arthritiε; and preventing MlP-driven transition from predominantly neutrophilic inflammation to the macrophage- dominant pathology which may be a key switch from acute, resolving inflammatory defenseε to chronic, ultimately destructive, mechanisms, e.g. auto-immune diseaεeε. Theεe antagoniεt/inhibitors may also be useful for treating silicosiε, sarcoidosis, idiopathic pulmonary -25- SUBST1TUTESHEET(RULE26) fibrosis and other chronic inflammatory diseases of the lung; idiopathic hyper-eoεinophilic syndrome since elevated MIP production exacerbates eosinophil production and migration; endotoxic εhock cauεed by elevated levelε of MIP in reεponεe to endotoxinε; hiεtamine-mediated allergic reactionε by inhibiting MlP-induced maεt cell and baεophil degranulation; inhibition of proεtaglandin-independent fever induced by MIP- lα; and preventing εuppressive effects of excess MlP-lα in aplastic anemia and other cases of bone marrow failure. The antibodieε would also be useful for prognostic applications and clinical screening to track disease progresεion and the efficacy of therapeutic intervention. Reεident macrophageε produce little or no MIP mRNA, diagnoεtic antibodies able to detect this would be uεeful indicatorε of auto-immune diεeaεe and chronic inflammatory and infective states. The antagonist/inhibitors may be employed in a compoεition with a pharmaceutically acceptable carrier, e.g., aε hereinabove described. The present invention will be further described with reference to the following examples; however, it is to be underεtood that the preεent invention iε not limited to εuch exampleε. All partε or amounts, unlesε otherwiεe εpecified, are by weight. In order to facilitate underεtanding of the following exampleε certain frequently occurring methodε and/or termε will be described. "Plaεmidε" are designated by a lower case p preceded and/or followed by capital letters and/or numbers. The starting plasmidε herein are either commercially available, publicly available on an unreεtricted basis, or can be constructed from available plasmidε in accord with published procedures. In addition, equivalent plasmids to those described are known in the art and will be apparent to the ordinarily skilled artisan. -26- SUBSnTUTESHEET(RULE26) "Digeεtion" of DNA refers to catalytic cleavage of the DNA with a restriction enzyme that acts only at certain εequenceε in the DNA. The variouε reεtriction enzymeε used herein are commercially available and their reaction conditions, cofactorε and other requirements were used as would be known to the ordinarily εkilled artiεan. For analytical purposes, typically 1 μg of plasmid or DNA fragment is used with about 2 units of enzyme in about 20 μl of buffer solution. For the purpose of isolating DNA fragments for plasmid conεtruction, typically 5 to 50 μg of DNA are digeεted with 20 to 250 units of enzyme in a larger volume. Appropriate buffers and εubεtrate amounts for particular restriction enzymes are specified by the manufacturer. Incubation times of about 1 hour at 37'C are ordinarily used, but may vary in accordance with the εupplier'ε inεtructionε. After digeεtion the reaction iε electrophoresed directly on a polyacrylamide gel to isolate the desired fragment. Size separation of the cleaved fragments is performed using 8 percent polyacrylamide gel described by Goeddel, D. et al . , Nucleic Acids Res., 8:4057 (1980). "Oligonucleotideε" referε to either a εingle stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands which may be chemically synthesized. Such synthetic oligonucleotides have no 5' phosphate and thuε will not ligate to another oligonucleotide without adding a phoεphate with an ATP in the preεence of a kinase. A synthetic oligonucleotide will ligate to a fragment that has not been dephosphorylated. "Ligation" refers to the procesε of forming phosphodiester bonds between two double stranded nucleic acid fragments (Maniatiε, T., et al.. Id., p. 146). Unless otherwise provided, ligation may be accomplished using known buffers and conditions with 10 unitε to T4 DNA ligase -27- SUBSΠTUTESHEET(RULE26) ("ligaεe") per 0.5 μg of approximately equimolar amounts of the DNA fragmentε to be ligated. Unleεε otherwiεe εtated, transformation was performed aε described in the method of Graham, F. and Van der Eb, A., Virology, 52:456-457 (1973). Example 1 Bacterial Expression and Purification of MIP-3 The DNA sequence encoding for MIP-3, ATCC # 75676 is initially amplified using PCR oligonucleotide primers corresponding to the 5' and sequences of the processed MIP-3 protein (minus the signal peptide sequence) and the vector sequenceε 3' to the MIP-3 gene. Additional nucleotideε correεponding to Bam HI and Xbal were added to the 5' and 3' sequences respectively. The 5' oligonucleotide primer has the sequence 5'-TCAGGATCCGTCACAAAAGATGCAGA-3' contains a BamHI restriction enzyme site followed by 18 nucleotides of MIP-3 coding sequence εtarting from the preεumed terminal amino acid of the proceεεed protein codon. The 3' sequence 3'-CGCTCTAGAGTAAAACGACGGCCAGT-5' contains complementary sequences to Xbal site and to a pBluescript SK- vector sequence located 3' to the MIP-3 DNA insert. The restriction enzyme sites correspond to the restriction enzyme sites on the bacterial expresεion vector PQE-9. (Qiagen, Inc. 9259 Eton Avenue, Chatεworth, CA, 91311). PQE-9 encodeε antibiotic resistance (-Amp r ) , a bacterial origin of replication (ori), an IPTG-regulatable promoter operator (P/0), a ribosome binding εite (RBS), a 6-Hiε tag and restriction enzyme sites. pQE-9 is then digested with BamHI and Xbal. The amplified sequences are ligated into PQE-9 and are inserted in frame with the sequence encoding for the histidine tag and the RBS. Figure 6 shows a schematic representation of this arrangement. The ligation mixture is then used to transform E. coli strain M15/rep4 available from Qiagen under the trademark M15/rep 4. M15/rep4 contains multiple copies of the plasmid pREP4, which expresses the -28- SUBST1TUTESHEET(RULE26) lacl represεor and also confers kanamycin resistance (Kan r ). Transformants are identified by their ability to grow on LB plates and ampicillin/kanamycin reεiεtant colonies are selected. Plasmid DNA is isolated and confirmed by restriction analyεiε. Cloneε containing the desired constructε are grown overnight (O/N) in liquid culture in LB media supplemented with both Amp (100 ug/ml) and Kan (25 ug/ml) . The O/N culture is used to inoculate a large culture at a ratio of 1:100 to 1:250. The cells are grown to an optical density 600 (O.D. 600 ) of between 0.4 and 0.6. IPTG ( "Isopropyl-B-D-thiogalacto pyranoside") is then added to a final concentration of 1 mM. IPTG induces by inactivating the lacl repressor, clearing the P/O leading to increaεed gene expression. Cells are grown an extra 3 to 4 hourε. Cells are then harvested by centrifugation. The cell pellet is solubilized in the chaotropic agent 6 Molar Guanidine HCl. After clarification, εolubilized MIP-3 iε purified from thiε εolution by chromatography on a Nickel-Chelate column under conditionε that allow for tight binding by proteinε containing the 6-Hiε tag. Hochuli, E. et al., J. Chromatography 411:177-184 (1984). MIP-3 (95% pure) is eluted from the column in 6 molar guanidine HCl pH 5.0 and for the purpose of renaturation adjusted to 3 molar guanidine HCl, lOO M sodium phosphate, 10 mmolar glutathione (reduced) and 2 mmolar glutathione (oxidized). After incubation in thiε εolution for 12 hours the protein iε dialyzed to 10 mmolar sodium phosphate. The presence of a new protein corresponding to 14 kd following induction demonstrated expression of the MIP-3. Example 2 Bacterial Expression and Purification of MIP-4 The DNA sequence encoding for MIP-4 ATCC # 75675 was initially amplified using PCR oligonucleotide primers corresponding to the 5' sequenceε of the processed MIP-4 -29- SUBSΠTUTE SHEET (RULE protein (minus the signal peptide sequence) and sequences in the pBSK vector 3' to MIP-4 gene. Additional nucleotides correεponding to Bam HI and Xbal were added to the 5' and 3' sequences respectively. The 5' oligonucleotide primer has the sequence TCAGGATCCTGTGCACAAGTTGGTACC contains a BamHI restriction enzyme site followed by 18 nucleotideε of MIP-4 coding εequence εtarting from the presumed terminal amino acid of the procesεed protein codon; The 3' εequence CGCTCTAGAGTAAAACGACGGCCAGT containε complementary εequenceε to Xbal site and to a pBluescript SK- vector εequence located 3' to the MIP-4 DNA insert. The restriction enzyme siteε correspond to the restriction enzyme εiteε on the bacterial expreεεion vector pQE-9 (Qiagen, Inc. 9259 Eton Avenue, Chatεworth, CA, 91311). pQE-9 encodes antibiotic resiεtance (Amp r ) , a bacterial origin of replication (ori), an IPTG- regulatable promoter operator (P/O), a riboεome binding site (RBS), a 6-His tag and reεtriction enzyme sites. pQE-9 was then digested with BamHI and Xbal The amplified sequences were ligated into pQE-9 and were inserted in frame with the εequence encoding for the hiεtidine tag and the RBS. Figure 6 εhowε a schematic repreεentation of thiε arrangement. The ligation mixture waε then used to transform E. coli εtrain available from Qiagen under the trademark M15/rep 4. M15/rep4 containε multiple copies of the plasmid pREP4, which expreεseε the lacl repreεεor and also confers kanamycin resiεtance (Kan r ) . Tranεformantε are identified by their ability to grow on LB plateε and ampicillin/kanamycin reεiεtant colonieε were εelected. Plaεmid DNA waε iεolated and confirmed by restriction analysis. Clones containing the desired constructε were grown overnight (O/N) in liquid culture in LB media εupplemented with both Amp (100 ug/ml) and Kan (25 ug/ml). The O/N culture iε uεed to inoculate a large culture at a ratio of 1:100 to 1:250. The cellε were grown to an optical denεity 600 (O.D. 600 ) of between 0.4 and 0.6. IPTG ("Isopropyl-B-D-thiogalacto pyranoside") was then -30- SUBSΠTUTESHEET(RULE26) added to a final concentration of 1 mM. IPTG induces by inactivating the lacl repressor, clearing the P/O leading to increaεed gene expression. Cells were grown an extra 3 to 4 hours. Cells were then harvested by centrifugation. The cell pellet waε εolubilized in the chaotropic agent 6 Molar Guanidine HCl. After clarification, εolubilized MIP-4 waε purified from thiε εolution by chromatography on a Nickel- Chelate column under conditionε that allow for tight binding by proteins containing the 6-His tag. Hochuli, E. et al., J. Chromatography 411:177-184 (1984). MIP-4 (95% pure) was eluted from the column in 6 molar guanidine HCl pH 5.0 and for the purpose of renaturation adjusted to 3 molar guanidine HCl, lOOmM sodium phosphate, 10 mmolar glutathione (reduced) and 2 mmolar glutathione (oxidized) . After incubation in this εolution for 12 hours the protein was dialyzed to 10 mmolar sodium phosphate. The presence of a new protein corresponding to 14 kd following induction demonstrated expression of the MIP-4. (Figure 5). Example 3 Expression of Recombinant MIP—3 in COS cells The expression of plasmid, CMV-MIP-3 HA is derived from a vector pcDNAl/Amp (Invitrogen) containing: 1) SV40 origin of replication, 2) ampicillin reεistance gene, 3) E.coli replication origin, 4) CMV promoter followed by a polylinker region, a SV40 intron and polyadenylation site. A DNA fragment encoding the entire MIP-3 precurεor and a HA tag fused in frame to its 3' end iε cloned into the polylinker region of the vector, therefore, the recombinant protein expression is directed under the CMV promoter. The HA tag correεpond to an epitope derived from the influenza hemagglutinin protein aε previouεly deεcribed (I. Wilson, H. Ni an, R. Heighten, A Cherenson, M. Connolly, and R. Lerner, 1984, Cell 37, 767). The infusion of HA tag to our target -31- SUBST1TUTESHEET(RULE26) protein allowε easy detection of the recombinant protein with an antibody that recognizes the HA epitope. The plaεmid conεtruction εtrategy iε described as follow: The DNA sequence, ATCC # 75676, encoding for MIP-3 is constructed by PCR on the original EST cloned using two primers: the 5' primer (5'GGAAAGCTTATGAAGGTCTCCGTGGCT-3' ) contains a HindiII εite followed by 18 nucleotideε of MIP-3 coding sequence starting from the initiation codon; the 3' sequence ( 5 '-CGCTCTAGATCAAGCGTAGTCTGGGACGTCGTA- TGGGTAATTCTTCCTGGTCTTGATCC -3' ) contains complementary sequenceε to Xba I site, translation stop codon, HA tag and the last 20 nucleotideε of the MIP-3 coding εequence (not including the εtop codon) . Therefore, the PCR product contains a Hindlll site, MIP-3 coding sequence followed by HA tag fused in frame, a translation termination stop codon next to the HA tag, and an Xbal site. The PCR amplified DNA fragment and the vector, pcDNAl/Amp, are digeεted with HindiII and Xbal restriction enzyme and ligated. The ligation mixture is transformed into E. coli strain SURE (available from Stratagene Cloning Syεtemε, 11099 North Torrey Pines Road, La Jolla, CA 92037) the transformed culture iε plated on ampicillin media plateε and reεiεtant colonieε are selected. Plasmid DNA is isolated from transformantε and examined by restriction analyεiε for the presence of the correct fragment. For expresεion of the recombinant MIP-3, COS cellε are tranεfected with the expresεion vector by DEAE- DEXTRAN method. (J. Sambrook, E. Fritεch, T. Maniatiε, Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Preεε, (1989)). The expression of the MIP-3-HA protein is detected by radiolabelling and immunoprecipitation method. (E. Harlow, D. Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, (1988)). Cells are labelled for 8 hourε with 35 S-cyεteine two days post tranεfection. Culture media are then collected and cellε are lysed with detergent (RIPA buffer (150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50mM Tris, pH 7.5). (Wilson, I. et al.. Id. 37:767 (1984)). Both cell lyεate and culture media are precipitated with a HA specific monoclonal antibody. Proteins precipitated are analyzed on 15% SDS-PAGE gels. Example 4 Expression of Recombinant MIP-4 in COS cells The expresεion of plaεmid, CMV-MIP-4 HA iε derived from a vector pcDNAl/Amp (Invitrogen) containing: 1) SV40 origin of replication, 2) ampicillin reεiεtance gene, 3) E.coli replication origin, 4) CMV promoter followed by a polylinker region, a SV40 intron and polyadenylation εite. A DNA fragment encoding the entire MIP-4 precurεor and a HA tag fused in frame to its 3' end is cloned into the polylinker region of the vector, therefore, the recombinant protein expression is directed under the CMV promoter. The HA tag correspond to an epitope derived from the influenza hemagglutinin protein as previously described (I. Wilson, H. Ni an, R. Heighten, A Cherenson, M. Connolly, and R. Lerner, 1984, Cell 37, 767). The infuεion of HA tag to our target protein allowε eaεy detection of the recombinant protein with an antibody that recognizeε the HA epitope. The plaεmid conεtruction εtrategy iε deεcribed aε follow: The DNA sequence ATCC # 75675 encoding for MIP-4 is constructed by PCR on the original EST cloned uεing two primerε: the 5' primer (5' GGAAAGCTTATGAAGGGCCTTGCAGCTGCC 3') containε a Hindlll εite followed by 20 nucleotides of MIP-4 coding sequence εtarting from the initiation codon; the 3 ' ε e q u e n c e ( 5 ' CGCTCTAGATCAABCGTAGTCTGGGACGTCGTATGGGTAGGCATTCAGCTTCAGGTC 3') contains complementary sequences to Xba I site, translation stop codon, HA tag and the last 19 nucleotideε of the MIP-4 -33- SUBST1TUTESHEET(RULE26) coding sequence (not including the stop codon) . Therefore, the PCR product contains a HindiII site, MIP-4 coding sequence followed by HA tag fused in frame, a translation termination stop codon next to the HA tag, and an Xbal εite. The PCR amplified DNA fragment and the vector, pcDNAl/Amp, are digeεted with HindiII and Xbal restriction enzyme and ligated. The ligation mixture iε tranεformed into E. coli strain SURE (available from Stratagene Cloning Systems, 11099 North Torrey Pines Road, La Jolla, CA 92037) the transformed culture is plated on ampicillin media plates and reεiεtant colonies are εelected. Plaεmid DNA iε iεolated from transformants and examined by reεtriction analyεiε for the preεence of the correct fragment. For expreεsion of the recombinant MIP-4, COS cells are tranεfected with the expreεεion vector by DEAE-DEXTRAN method. (J. Sambrook, E. Fritsch, T. Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Presε, (1989)). The expreεεion of the MIP-4-HA protein iε detected by radiolabelling and immunoprecipitation method. (E. Harlow, D. Lane, Antibodieε: A Laboratory Manual, Cold Spring Harbor Laboratory Press, (1988)). Cells are labelled for 8 hourε with 35 S-cyεteine two dayε poεt tranεfection. Culture media are then collected and cellε are lysed with detergent (RIPA buffer (150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50mM Tris, pH 7.5). (Wilson, I. et al.. Id. 37:767 (1984)). Both cell lyεate and culture media are precipitated with a HA specific monoclonal antibody. Proteinε precipitated are analyzed on 15% SDS-PAGE gels. Example 5 Expression pattern of MIP-3 in human tiεεue Northern blot analysiε waε carried out to examine the levels of expresεion of MIP-3 in human tissues. Total cellular RNA samples were isolated with RNAzol™ B system (Biotecx Laboratories, Inc. 6023 South Loop East, Houston, TX -34- SUBSTΓRJTESHEET(RULE26) 77033). About lOug of total RNA isolated from each human tiεεue εpecified iε εeparated on 1% agaroεe gel and blotted onto a nylon filter. (Sambrook, Fritsch, and Maniatis, Molecular Cloning, Cold Spring Harbor Presε, (1989)). The labeling reaction is done according to the Stratagene Prime- It kit with 50ng DNA fragment. The labeled DNA iε purified with a Select-G-50 column. (5 Prime - 3 Prime, Inc. 5603 Arapahoe Road, Boulder, CO 80303). The filter iε then hybridized with radioactive labeled full length MIP-3 gene at 1,000,000 cpm/ml in 0.5 M NaP0 4 , pH 7.4 and 7% SDS overnight at 65°C. After wash twice at room temperature and twice at 60"C with 0.5 x SSC, 0.1% SDS, the filter is then exposed at -70'C overnight with an intensifying screen. Example 6 Expression pattern of MIP-4 in human cells Northern blot analysiε waε carried out to examine the levelε of expreεεion of MIP-4 in human cellε. Total cellular RNA sampleε were iεolated with RNAzol™ B εyεtem (Biotecx Laboratorieε, Inc. 6023 South Loop East, Houston, TX 77033). About lOug of total RNA isolated from each human tissue specified was separated on 1% agarose gel and blotted onto a nylon filter. (Sambrook, Fritεch, and Maniatiε, Molecular Cloning, Cold Spring Harbor Press, (1989)). The labeling reaction waε done according to the Stratagene Prime-It kit with 50ng DNA fragment. The labeled DNA waε purified with a Select-G-50 column. (5 Prime - 3 Prime, Inc. 5603 Arapahoe Road, Boulder, CO 80303). The filter waε then hybridized with radioactive labeled full length MIP-4 gene at 1,000,000 cpm/ml in 0.5 M NaP0 4 , pH 7.4 and 7% SDS overnight at 65 * C. After wash twice at room temperature and twice at 60'C with 0.5 x SSC, 0.1% SDS, the filter waε then exposed at -70 * C overnight with an intensifying screen. See Figure 6. Example 7 -35- SUBST1TUTESHEET(RULE26) Bacterial Expression and Purification of MIP-I7 The DNA sequence encoding for MIP-I7, PBLSKMIP (ATCC # 75572) is initially amplified using PCR oligonucleotide primers corresponding to the 5 ' and 3 ' sequences of the procesεed MIP-I7 protein (minuε the signal peptide sequence) and additional nucleotides correεponding to Bam HI and Xbal were added to the 5' and 3' εequenceε reεpectively. The 5' oligonucleotide primer haε the εequence GCCCGCGGATCCTCCTCACGGGGACCTTAC containε a BamHI reεtriction enzyme εite followed by 15 nucleotideε of MIP-I7 coding εequence εtarting from the preεumed terminal amino acid of the processed protein codon; The 3' sequence GCCTGCTCTAGATCAAAGCAGGGAAGCTCCAG contains complementary sequenceε to Xbal site a translation stop codon and the last 20 nucleotides of MIP-I7 coding sequence. The restriction enzyme εiteε correεpond to the restriction enzyme εiteε on the bacterial expreεεion vector PQE-9. (Qiagen, Inc. 9259 Eton Avenue, Chatεworth, CA, 91311). PQE-9 encodeε antibiotic reεiεtance (Amp r ) , a bacterial origin of replication (ori) , an IPTG-regulatable promoter operator (P/O), a riboεome binding εite (RBS), a 6-Hiε tag and reεtriction enzyme sites. pQE-9 was then digested with BamHI and Xbal The amplified εequenceε were ligated into PQE-9 and were inεerted in frame with the εequence encoding for the hiεtidine tag and the RBS. Figure 6 εhowε a εchematic repreεentation of thiε arrangement. The ligation mixture waε then uεed to tranεform E. coli εtrain available from Qiagen under the trademark M15/rep 4. M15/rep4 containε multiple copieε of the plaεmid pREP4, which expresses the lacl represεor and alεo conferε kanamycin reεiεtance (Kan r ). Tranεformantε are identified by their ability to grow on LB plateε and ampicillin/kanamycin reεiεtant colonieε were εelected. Plaεmid DNA was isolated and confirmed by reεtriction analysis. Clones containing the desired constructε were grown overnight (O/N) in liquid culture in LB -36- SUBSTTTUTESHEET(RULE26) media supplemented with both Amp (100 ug/ml) and Kan (25 ug/ml). The O/N culture is used to inoculate a large culture at a ratio of 1:100 to 1:250. The cells were grown to an optical denεity 600 (O.D. 600 ) of between 0.4 and 0.6. IPTG ("Iεopropyl-B-D-thiogalacto pyranoεide") waε then added to a final concentration of 1 mM. IPTG induces by inactivating the lad repreεεor, clearing the P/O leading to increased gene expression. Cells were grown an extra 3 to 4 hours. Cells were then harvested by centrifugation. The cell pellet was εolubilized in the chaotropic agent 6 Molar Guanidine HCl. After clarification, εolubilized MIP-I7 waε purified from thiε εolution by chromatography on a Nickel-Chelate column under conditions that allow for tight binding by proteins containing the 6-His tag. Hochuli, E. et al., J. Chromatography 411:177-184 (1984). MIP-I7 (95% pure) waε eluted from the column in 6 molar guanidine HCl pH 5.0 and for the purpoεe of renaturation adjusted to 3 molar guanidine HCl, lOOmM sodium phosphate, 10 mmolar glutathione (reduced) and 2 mmolar glutathione (oxidized) . After incubation in this solution for 12 hours the protein waε dialyzed to 10 mmolar εodium phosphate. The preεence of a new protein correεponding to 14 kd following induction demonεtrated expreεεion of the MIP-I7. (Figure 3). -37- SUBST1TUTESHEET(RULE26) Example 8 Expression of Recombinant MIP-I7 in COS cells The expresεion of plasmid, CMV-MIP-1 7 HA is derived from a vector pcDNAI/Amp (Invitrogen) containing: 1) SV40 origin of replication, 2) ampicillin resistance gene, 3) E.coli replication origin, 4) CMV promoter followed by a polylinker region, a SV40 intron and polyadenylation site. A DNA fragment encoding the entire MIP-1 7 precursor and a HA tag fused in frame to itε 3 ' end waε cloned into the polylinker region of the vector, therefore, the recombinant protein expreεεion iε directed under the CMV promoter. The HA tag correεpond to an epitope derived from the influenza hemagglutinin protein aε previouεly deεcribed (I. Wilεon, H. Niman, R. Heighten, A Cherenson, M. Connolly, and R. Lerner, 1984, Cell 37, 767). The infusion of HA tag to our target protein allows eaεy detection of the recombinant protein with an antibody that recognizeε the HA epitope. The plasmid construction εtrategy iε deεcribed aε follow: The DNA εequence, pBLSKMIP (ATCC # 75572), encoding for MIP-I7 waε conεtructed by PCR on the original EST cloned using two primers: the 5' primer (5'GGAAAGCTTATGAAGATTCCGTGGCTGC- 3') contains a Hindlll site followed by 20 nucleotides of MIP- 1 7 coding sequence starting from the initiation codon; the 3' sequence (5'-CGC'lC'-l-AGATCAAGCX^ TTG -3') containε complementary εequenceε to Xba I εite, tranεlation εtop codon, HA tag and the laεt 19 nucleotideε of the MIP-1 7 coding εequence (not including the εtop codon) . Therefore, the PCR product containε a Hindlll εite, MIP-1 7 coding εequence followed by HA tag fused in frame, a tranεlation termination εtop codon next to the HA tag, and an Xbal site. The PCR amplified DNA fragment and the vector, pcDNAI/Amp, were digested with Hindlll and Xbal restriction enzyme and ligated. The ligation mixture was transformed into E. coli εtrain SURE (available from Stratagene Cloning Systems, 11099 North Torrey Pines Road, La Jolla, CA 92037) the transformed culture was plated on ampicillin media plates and resistant colonies were selected. Plasmid DNA was isolated from transformantε and examined by restriction analysiε for the preεence of the correct fragment. For expreεεion of the recombinant MIP-I7, COS cellε were transfected with the expresεion vector by DEAE-DEXTRAN method. (J. Sambrook, E. Fritεch, T. Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Preεε, (1989)). The expreεεion of the MIP-1 7-HA protein was detected by radiolabelling and immunoprecipitation method. (E. Harlow, D. Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, (1988)). Cells were labelled for 8 hours with 35 S-cysteine two days post transfection. Culture media were then collected and cells were lysed with detergent (RIPA buffer (150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50mM Tris, pH 7.5). (Wilson, I. et al.. Id. 37:767 (1984)). Both cell lysate and culture media were precipitated with a HA specific monoclonal antibody. Proteinε precipitated were analyzed on 15% SDS-PAGE gelε. Example 9 Expression pattern of MIP-I7 in human tisεue Northern blot analysiε waε carried out to examine the levelε of expreεεion of MIP-l in human tissueε. Total cellular RNA εampleε were iεolated with RNAzol™ B εyεtem (Biotecx Laboratorieε, Inc. 6023 South Loop Eaεt, Houεton, TX 77033). About lOug of total RNA iεolated from each human tissue specified was separated on 1% agarose gel and blotted onto a nylon filter. (Sambrook, Fritsch, and Maniatis, Molecular Cloning, Cold Spring Harbor Press, (1989)). The labeling reaction was done according to the Stratagene Prime- It kit with 50ng DNA fragment. The labeled DNA was purified with a Select-G-50 column. (5 Prime - 3 Prime, Inc. 5603 Arapahoe Road, Boulder, CO 80303). The filter was then hybridized with radioactive labeled full length MIP-l gene at 1,000,000 cpm/ml in 0.5 M NaP0 4 , pH 7.4 and 7% SDS overnight at 65 * C. After waεh twice at room temperature and twice at 60 * C with 0.5 x SSC, 0.1% SDS, the filter waε then expoεed at -70"C overnight with an intenεifying εcreen. The message RNA for MlP-lpis abundant in spleen, lung, liver and less so in other tiεεueε. (Figure 5). Numerouε modificationε and variationε of the preεent invention are poεεible in light of the above teachingε and, therefore, within the εcope of the appended claimε, the invention may be practiced otherwiεe than as particularly deεcribed. SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: LI, ET AL. (ii) TITLE OF INVENTION: Macrophage Inflammatory Protein -3, -4 and 1 Gamma (iii) NUMBER OF SEQUENCES: 6 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: CARELLA, BYRNE, BAIN, GILFILLAN, CECCHI, STEWART & OLSTEIN (B) STREET: 6 BECKER FARM ROAD (C) CITY: ROSELAND (D) STATE: NEW JERSEY (E) COUNTRY: USA (F) ZIP: 07068 (V) COMPUTER READABLE FORM: (A) MEDIUM TYPE: 3.5 INCH DISKETTE (B) COMPUTER: IBM PS/2 (C) OPERATING SYSTEM: MS-DOS (D) SOFTWARE: WORD PERFECT 5.1 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE: (C) CLASSIFICATION: (vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: 08/173,209 (B) FILING DATE: 22-DEC-93 (viii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: 08/208,339 (B) FILING DATE: 08-MAR-94 (ix) ATTORNEY/AGENT INFORMATION: (A) NAME: FERRARO, GREGORY D. (B) REGISTRATION NUMBER: 36,134 (C) REFERENCE/DOCKET NUMBER: 325800-163 (x) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 201-994-1700 (B) TELEFAX: 201-994-1744 (2) INFORMATION FOR SEQ ID NO:l: (i) SEQUENCE CHARACTERISTICS (A) LENGTH: 474 BASE PAIRS (B) TYPE: NUCLEIC ACID (C) STRANDEDNESS: SINGLE (D) TOPOLOGY: LINEAR (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: TGAAGCTCCC ACCAGGCCAG CTCTCCTCCC ACAACAGCTT CCCACAGCAT GAAGATCTCC 60 GTGGCTGCAA TTCCCTTCTT CCTCCTCATC ACCATCGCCC TAGGGACCAA GACTGAATCC 120 TCCTCACGGG GACCTTACCA CCCCTCAGAG TGCTGCTTCA CCTACACTAC CTACAAGATC 180 CCGCGTCAGC GGATTATGGA TTACTATGAG ACCAACAGCC AGTGCTCCAA GCCCGGAATT 240 GTCTTCATCA CCAAAAGGGG CCATTCCGTC TGTACCAACC CCAGTGACAA GTGGGTCCAG 300 GACTATATCA AGGACATGAA GGAGAACTGA GTGACCCAGA AGGGGTGGCG AAGGCACAGC 360 TCAGAGACAT AAAGAGAAGA TGCCAAGGCC CCCTCCTCCA CCCACCGCTA ACTCTCAGCC 420 CCAGTCACCC TCTTGGAGCT TCCCTGCTTT GAATTAAAGA CCACTCATGC TCTT 474 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS (A) LENGTH: 93 AMINO ACIDS (B) TYPE: AMINO ACID (C) STRANDEDNESS: (D) TOPOLOGY: LINEAR (ii) MOLECULE TYPE: PROTEIN ( i) SEQUENCE DESCRIPTION: SEQ ID NO:2: Met Lys lie Ser Val Ala Ala lie Pro Phe Phe Leu Leu lie Thr -20 -15 -10 lie Ala Leu Gly Thr Lys Thr Glu Ser Ser Ser Arg Gly Pro Tyr -5 1 5 His Pro Ser Glu Cys Cys Phe Thr Tyr Thr Thr Tyr Lys lie Pro 10 15 20 Arg Gin Arg lie Met Asp Tyr Tyr Glu Thr Asn Ser Gin Cys Ser 25 30 35 Lys Pro Gly lie Val Phe lie Thr Lys Arg Gly His Ser Val Cys 40 45 50 Thr Asn Pro Ser Asp Lys Trp Val Gin Asp Tyr lie Lys Asp Met 55 60 65 Lys Glu Asn (2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS (A) LENGTH: 366 BASE PAIRS (B) TYPE: NUCLEIC ACID (C) STRANDEDNESS: SINGLE (D) TOPOLOGY: LINEAR (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: ATGAAGGTCT CCGTGGCTGC CCTCTCCTGC CTACATGCCT TTTTACTGCC CTTGGTCCCA 60 GGGCCGGGTC ACAAAAGATG CAGAGACAGA GTTCATGAAT GTCAAAGCTT CATTGGAAAG 120 -43- SUBSΠTUTESHEET(RULE26) ATCCAGTACT TCTGGGACAG ATTCCATGCT ACTAGTGCTG ACTGCTGCAT CTCCTACACC 180 CCACGAAGCA TCCCGTGTTC ACTCCTGGAG AGTTACTTTG AAACGAACAG CGAGTGCTCC 240 AAGCCGGGTG TCATCTTCCT CACCAAGAAG GGGCGACGTT TCTGTGCCAA CCCCAGTGAT 300 AAGCAAGTTC AGGTTTGCAT GAGAATGCTG AAGCTGGACA CACGGATCAA GACCAGGAAG 360 AATTGA 366 (2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS (A) LENGTH: 121 AMINO ACIDS (B) TYPE: AMINO ACID (C) STRANDEDNESS: (D) TOPOLOGY: LINEAR (ii) MOLECULE TYPE: PROTEIN (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: Met Lys Val Ser Val Ala Ala Leu Ser Cys Leu His Ala Phe Leu -45 -40 -35 Leu Pro Leu Val Pro Gly Pro Gly His Lys Arg Cys Arg Asp Arg -30 -25 -20 Val His Glu Cys Gin Ser Phe His Trp Lys lie Gin Tyr Phe Trp -15 -10 -5 Asp Arg Phe His Ala Thr Ser Ala Asp Cys Cys lie Ser Tyr Thr 5 10 15 Pro Arg Ser lie Pro Cys Ser Leu Leu Gin Ser Tyr Phe Glu Thr 20 25 30 Asn Ser Glu Cys Ser Lys Pro Gly Val lie Phe Leu Thr Lys Lys 35 40 45 Gly Arg Arg Phe Cys Ala Asn Pro Ser Asp Lys Gin Val Gin Val 50 55 60 Cys Met Arg Met Leu Lys Leu Asp Thr Arg lie Lys Thr Arg Lys 65 70 75 Asn (3) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS (A) LENGTH: 270 BASE PAIRS (B) TYPE: NUCLEIC ACID (C) STRANDEDNESS: SINGLE (D) TOPOLOGY: LINEAR (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: ATGAAGGGCC TTGCAGCTGC CCTCCTTGTC CTCGTCTGCA CCATGGCCCT CTGCTCCTGT 60 GCACAAGTTG GTACCAACAA AGAGCTCTGC TGCCTCGTCT ATACCTCCTG GCAGATTCCA 120 CAAAAGTTCA TAGTTGACTA TTCTGAAACC AGCCCCCAGT GCCCCAAGCC AGGTGTCATC 180 CTCCTAACCA AGAGAGGCCG GCAGATCTGT GCTGACCCCA ATAAGAAGTG GGTCCAGAAA 240 TACATCAGCG ACCTGAAGCT GAATGCCTGA 270 (4) INFORMATION FOR SEQ ID NO:6: (i) SEQUENCE CHARACTERISTICS (A) LENGTH: 89 AMINO ACIDS (B) TYPE: AMINO ACID (C) STRANDEDNESS: (D) TOPOLOGY: LINEAR (ii) MOLECULE TYPE: PROTEIN (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Met Lys Gly Leu Ala Ala Ala Leu Leu Val Leu Val Cys Thr Met -15 -10 -5 Ala Leu Cys Ser Cys Ala Gin Val Gly Thr Asn Lys Glu Leu 5 10 Cys Cys Leu Val Tyr Thr Ser Trp Gin lie Pro Gin Lys Phe lie 15 20 25 -45- SUBSπTUTESHEET(RULE26) Val Asp Tyr Ser Glu Thr Ser Pro Gin Cys Pro Lys Pro Gly Val 30 35 40 lie Leu Leu Thr Lys Arg Gly Arg Gin lie Cys Ala Asp Pro Asn 45 50 55 Lys Lys Trp Val Gin Lys Tyr lie Ser Asp Leu Lys Leu Asn Ala 60 65 70 -46- SUBSTtTUTESHEET(RULE26)

Claims: WHAT IS CLAIMED IS: 1. An isolated polynucleotide selected from the group consisting of (a) a polynucleotide encoding for the MIP-3 polypeptide having the deduced amino acid sequence of Figure 1 or a fragment, analog or derivative of said polypeptide; (b) a polynucleotide encoding for the MIP-3 polypeptide having the amino acid sequence encoded by the cDNA contained in ATCC Deposit No. 75676 or a fragment, analog or derivative of said polypeptide; (c) a polynucleotide encoding for the MIP-4 polypeptide having the deduced amino acid sequence of Figure 2 or a fragment, analog or derivative of said polypeptide; and (d) a polynucleotide encoding for the MIP-4 polypeptide having the amino acid sequence encoded by the cDNA contained in ATCC Deposit No. 75675 or a fragment, analog or derivative of said polypeptide. 2. The polynucleotide of Claim 1 wherein the polynucleotide is DNA. 3. The polynucleotide of Claim 1 wherein the polynucleotide is RNA. 4. The polynucleotide of Claim 1 wherein the polynucleotide is genomic DNA. 5. The polynucleotide of Claim 2 wherein said polynucleotide encodes for MIP-3 having the deduced amino acid sequence of Figure 1. 6. The polynucleotide of Claim 2 wherein said polynucleotide encodes for the MIP-3 polypeptide encoded by the cDNA of ATCC Deposit No. 75676. 7. The polynucleotide of Claim 2 wherein said polynucleotide encodes for MIP-4 having the deduced amino acid sequence of Figure 2. 8. The polynucleotide of Claim 2 wherein said polynucleotide encodes for the MIP-4 polypeptide encoded by the cDNA of ATCC Deposit No. 75675. 9. The polynucleotide of Claim 1 having the coding sequence for MIP-3 aε shown in Figure 1. 10. The polynucleotide of Claim 2 having the coding sequence for MIP-3 deposited as ATCC Deposit No. 75676. 11. The polynucleotide of Claim 1 having the coding sequence for MIP-4 as shown in Figure 2. 12. The polynucleotide of Claim 2 having the coding sequence for MIP-4 deposited aε ATCC Deposit No. 75675 13. A vector containing the DNA of Claim 2. 14. A hoεt cell genetically engineered with the vector of Claim 13. 15. A process for producing a polypeptide comprising: expressing from the host cell of Claim 14 the polypeptide encoded by said DNA. 16. A process for producing cells capable of expressing a polypeptide comprising genetically engineering cells with the vector of Claim 13. 17. An isolated DNA hybridizable to the DNA of Claim 2 and encoding a polypeptide having MIP-3 activity. 18. An isolated DNA hybridizable to the DNA of Claim 2 and encoding a polypeptide having MIP-4 activity. 19. A polypeptide selected from the group consisting of (i) a MIP-3 polypeptide having the deduced amino acid sequence of Figure 1 and fragments, analogs and derivatives thereof (ii) a MIP-3 polypeptide encoded by the cDNA of ATCC Deposit No. 75676 and fragments, analogs and derivatives of said polypeptide (iii) a MIP-4 polypeptide having the deduced amino acid sequence of Figure 2 and fragments, analogs and derivatives thereof and (iv) a MIP-4 polypeptide encoded by the cDNA of ATCC Deposit No. 75675 and fragments, analogs and derivatives of said polypeptide. 20. The polypeptide of Claim 19 wherein the polypeptide is MIP-3 having the deduced amino acid sequence of Figure 1. 21. The polypeptide of Claim 19 wherein the polypeptide is MIP-4 having the deduced amino acid sequence of Figure 2. 22. An antibody against the MIP-3 polypeptide of claim 19. 23. An antibody against a MIP-3 receptor. 24. An antibody against a MIP-4 receptor. 25. An antibody against the MIP-4 polypeptide of claim 19. 26. An antagonist/inhibitor against the MIP-3 polypeptide of claim 19. 27. An antagonist/inhibitor against the MIP-4 polypeptide of claim 19. 28. A method for the treatment of a patient having need of MIP-3 comprising: administering to the patient a therapeutically effective amount of the polypeptide of claim 19. 29. A method for the treatment of a patient having need of MIP-4 comprising: administering to the patient a therapeutically effective amount of the polypeptide of claim 19. 30. A method for the treatment of a patient having need to inhibit MIP-3 comprising: administering to the patient a therapeutically effective amount of an antagonist/inhibitor Claim 26. 31. A method for the treatment of a patient having need to inhibit MIP-4 comprising: administering to the patient a therapeutically effective amount of an antagonist/inhibitor Claim 27. 32. A pharmaceutical composition comprising the MIP-3 polypeptide of Claim 19 and a pharmaceutically acceptable carrier. 33. A pharmaceutical composition comprising the MIP-4 polypeptide of Claim 19 and a pharmaceutically acceptable carrier. 34. An isolated polynucleotide selected from the groups consisting of: (a) a polynucleotide encoding a MIP-I7 polypeptide having the deduced amino acid sequence of Figure 8 or a fragment, analog or derivative of said polypeptide; (b) a polynucleotide encoding a MIP-I7 polypeptide having the amino acid sequence encoded by the cDNA contained in ATCC Deposit No. 75572 or a fragment, analog or derivative of said polypeptide. 35. The polynucleotide of Claim 34 wherein the polynucleotide is DNA. 36. The polynucleotide of Claim 34 wherein the polynucleotide is RNA. 37. The polynucleotide of Claim 34 wherein the polynucleotide is genomic DNA. 38. The polynucleotide of Claim 35 wherein said polynucleotide encodes MIP-I7 having the deduced amino acid sequence of Figure 8. 39. The polynucleotide of Claim 35 wherein said polynucleotide encodes the MIP-I7 polypeptide encoded by the cDNA of ATCC Deposit No. 75572. 40. The polynucleotide of Claim 34 having the coding sequence of MIP-I7 as shown in Figure 8. 41. The polynucleotide of Claim 35 having the coding sequence of MIP-I7 deposited aε ATCC Deposit No. 75572. 42. A vector containing the DNA of Claim 35. 43. A host cell genetically engineered with the vector of Claim 42. 44. A process for producing a polypeptide comprising: expressing from the host cell of Claim 43 the polypeptide encoded by said DNA. 45. A process for producing cells capable of expressing a polypeptide comprising genetically engineering cells with the vector of Claim 42. 46. An isolated DNA hybridizable to the DNA of Claim 35 and encoding a polypeptide having MIP-I7 activity. 47. A polypeptide selected from the group consisting of (i) a MIP-I7 polypeptide having the deduced amino acid sequence of Figure 8 and fragments, analogs and derivatives thereof and (ii) a MIP-I7 polypeptide encoded by the cDNA of ATCC Deposit No. 75572 and fragments, analogs and derivatives of said polypeptide. 48. The polypeptide of Claim 14 wherein the polypeptide iε MIP- I having the deduced amino acid sequence of Figure 8. 49. An antibody against the polypeptide of claim 47. 50. An antagonist/inhibitor against the polypeptide of claim 47. 51. A method for the treatment of a patient having need of MIP- I7 comprising: administering to the patient a therapeutically effective amount of the polypeptide of claim 47. 52. A method for the treatment of a patient having need to inhibiit MIP-I7 comprising: administering to the patient a therapeutically effective amount of the antagonist/inhibitor of Claim 50. 53. A pharmaceutical composition comprising the polypeptide of Claim 47 and a pharmaceutically acceptable carrier. 54. The method of Claim 18 wherein said therapeutically effective amount of the polypeptide is administered by providing to the patient DNA encoding said polypeptide and expressing said polypeptide in vivo .

Inventors: ADAMS MARK D, LI HAODONG, ROSEN CRAIG A, RUBEN STEVE, ADAMS, MARK, D., LI, HAODONG, ROSEN, CRAIG, A., RUBEN, STEVE
Applicants: HUMAN GENOME SCIENCES INC, HUMAN GENOME SCIENCES, INC.
Application year: 1994
Main classification: A01N37
IPC codes: A01N37, A61K38, A61K48, A61P7, A61P9, A61P11, A61P17, A61P29, A61P31, A61P35, A61P37, A61P43, C07H21, C07K14, C07K16, C12N1, C12N5, C12N15, C12P21, C12Q1, C12R1